The present invention is in the field of phosphodiesterase proteins that are related to cGMP-stimulated 3xe2x80x2,5xe2x80x2-cyclic nucleotide phosphodiesterase 2A (PDE2A), recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods. In particular, the phosphodiesterase protein provided by the present invention is a novel alternative splice form of PDE2A.
Phosphodiesterases
In general, phosphodiesterases (xe2x80x9cPDEsxe2x80x9d) catalyze the hydrolysis of a phosphodiester bond. Specific classes of phosphodiesterases include those catalyzing the degradation of cyclilc monophosphates.
The signaling pathways regulated by PDEs include the transduction of photon capture in the outer segment of a photoreceptor as well as changes in neurotransmitter release from its inner segment. PDEs also regulate the aldosterone production by atrial natriuretic peptide and platelet aggregation by endothelial relaxation factor.
Experimental data have demonstrated the role of phosphodiesterases in a range of diseases, including inflammatory diseases such as asthma, chronic obstructive pulmonary disease, rheumatoid arthritis and atopy. Drugs that selectively inhibit individual PDE isozymes have a wide variety of different effects on an animals, suggesting specific roles for most of the different PDEs.
Experimental evidence indicates the existence of several related gene families coding for different phosphodiesterases, and that each of these families contain more than one gene. Furthermore, each gene product is differentially spliced in different tissues to yield different isozymes. Isolation of cDNAs for many of the isozymes has allowed a series of structure/function studies to be initiated. Several of these isozymes are regulated by phosphorylation/dephosphorylation mechanisms.
Over 30 phosphodiesterases have been identified. Categories of phosphodiesterases include seven major classes. Class I phosphodiesterases include calmodulin-dependent phosphodiesterases which are expressed in tissues such as the brain, testes, sperm, coronary artery, lung, heart, and pancreas. Class II phosphodiesterases include cGMP-stimulated phosphodiesterases which are expressed in tissues such as the brain, adrenal gland, and the heart. Class III phosphodiesterases include cGMP-inhibited phosphodiesterases expressed in tissues such as T-lymphocytes, macrophages, platelets, smooth muscle, heart, and adipose tissue. Class IV phosphodiesterases include cAMP-specific phosphodiesterases which are expressed in tissues such as monocytes, leukocytes, and the central nervous system. Class V phosphodiesterases include cGMP-specific phosphodiesterases which are expressed in tissues such as lung, smooth muscle, platelets, and the aorta. Class VI phosphodiesterases include photoreceptor-specific phosphodiesterases expressed in the retina. Class VII phosphodiesterases include high affinity cAMP-specific phosphodiesterases.
Cyclic Nucleotide Phosphodiesterases
As is well-known in the art, a myriad of physiological processes are controlling by causing changes in the steady state levels of the second messengers cAMP and cGMP. One of the major mechanisms by which these levels are controlled is via the cyclic nucleotide PDEs that control their degradation by catalyzing the hydrolysis of a phosphodiester bond, yielding 5xe2x80x2-AMP and 5xe2x80x2-GMP, respectively.
Experimental data have demonstrated the role of cyclic nucleotide phosphodiesterases in a range of diseases, including inflammatory diseases such as asthma, chronic obstructive pulmonary disease, rheumatoid arthritis and atopy.
In mammals, four genes are known to code for cAMP-specific PDEs. These genes are known as PDE4A, PDE4B, PDE4C and PDE4D. This was first demonstrated in rats and later in humans and in mice. The four human and four rat genes show a one to one correspondence, in that each of the four human PDE4 genes is more closely related to its homologous rat gene than to any other human gene. The PDE4 genes are located on three different human chromosomes: PDE4B on chromosome 1, PDE4D on chromosome 5; PDE4A on p13.2 of chromosome 19 and PDE4C on p13.1 of chromosome 19. Their four murine homologues are each located in correspondingly conserved regions of the mouse genome. The mammalian PDE4 genes thus comprise a well-conserved multigene family.
The existence of a large number of mRNA transcripts from many of the mammalian PDE4 genes suggests that the genomic structure of these genes is likely to be complex. Partial genomic sequences have been published for the rat PDE4B and PDE4D genes. However, the published data indicate that sequences at the 5xe2x80x2 end of the genes, which would include a number of upstream exons and promoter sites, were not included.
cGMP-stimulated 3xe2x80x2,5xe2x80x2-cyclic nucleotide phosphodiesterase 2A (PDE2A)
The novel human protein, and encoding gene, provided by the present invention is an alternative splice form of cGMP-stimulated 3xe2x80x2,5xe2x80x2-cyclic nucleotide phosphodiesterase 2A (PDE2A). Specifically, the phosphodiesterase provided by the present invention differs from known phosphodiesterases, particularly bovine PDE2A1 (gi116569) and human PDE2A3 (gi4505657), in exon 1. These difference are illustrated in the Figures, particularly in the amino acid sequence alignments shown in FIGS. 2 and 3.
For a further review of PDE2A and related proteins, see Rosman et al., Gene May 20, 1997;191(1):89-95; Sonnenburg et al., J Biol Chem Sep. 15, 1991;266(26):17655-61; Trong et al., Biochemistry Nov. 6, 1990;29(44):10280-8; and Charbonneau et al., Proc Natl Acad Sci U S A December 1986;83(24):9308-12.
Phosphodiesterase proteins, particularly alternative splice forms of PDE2A, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown splice forms of phosphodiesterase proteins. The present invention advances the state of the art by providing a previously unidentified human PDE2A alternative splice form.
The present invention is based in part on the identification of amino acid sequences of human phosphodiesterase peptides and proteins that are related to PDE2A, as well as allelic variants and other mammalian orthologs thereof. Specifically, the phosphodiesterase protein provided by the present invention is a novel alternative splice form of PDE2A. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate phosphodiesterase activity in cells and tissues that express the phosphodiesterase. Experimental data as provided in FIG. 1 indicates expression in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample.